Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.
Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.Show MeSH
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Mentions: We screened all multicopy transformants using a 96-well plate luminescence assay and selected the “brightest” strains (Figure 3B) for time-lapse luminescence microscopy. The variability in luminescence between transformants arises from unequal copy number integration in chromosomal loci due to homologous ends-in recombination. Time-lapse luminescence microscopy using standard, agarose pad methods initially exhibited bright luminescence signal. However, the signal disappeared within 15 min. We reasoned that luciferase substrates (d-luciferin, oxygen) were being depleted. Thus we combined a CellAsic microfluidic device (EMD Millipore, Billerica, MA) with time-lapse luminescence microscopy. The microfluidic device trapped yeast cells and maintained growth medium at constant levels through perfusion. The medium exchange time was <1 min, and cells were unable to deplete their substrates. With microfluidics, we consistently measured in vivo gene expression dynamics with subminute time resolution for >12 h (Figure 4). We only stopped time lapse because the yeast microcolony extended beyond the field of view. We further validated our method by benchmarking and quantifying gene induction and repression dynamics of many green, yellow, and red luciferase reporters for several metabolite-repressed yeast promoters (MET17, LEU1, ADE17, LYS9); see Supplemental Figures S2–S4 and Supplemental Table S1.
Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.